System for biomarker discovery

Abstract

The present application discloses a method for discovering a methylation marker gene for the conversion of a cell comprising: (i) comparing converted and unconverted cell gene expression content to identify a gene that is present in greater abundance in the unconverted cell; (ii) treating a converted cell with a demethylating agent and comparing its gene expression content with gene expression content of an untreated converted cell to identify a gene that is present in greater abundance in the cell treated with the demethylating agent; and (iii) identifying a gene that is common to the identified genes in steps (i) and (ii), wherein the common identified gene is the methylation marker gene.

1 . A method for discovering a methylation marker gene for the conversion of a cell comprising: 
 (i) comparing converted and unconverted cell gene expression content to identify a gene that is present in greater abundance in the unconverted cell;    (ii) treating a converted cell with a demethylating agent and comparing its gene expression content with gene expression content of an untreated converted cell to identify a gene that is present in greater abundance in the cell treated with the demethylating agent; and    (iii) identifying a gene that is common to the identified genes in steps (i) and (ii), wherein the common identified gene is the methylation marker gene.    
     
     
         2 . The method according to  claim 1 , wherein the comparing is carried out by direct comparison.  
     
     
         3 . The method according to  claim 1 , wherein the comparing is carried out by indirect comparison.  
     
     
         4 . The method according to  claim 1 , wherein the converted cell is cancer cell.  
     
     
         5 . The method according to  claim 4 , wherein the cancer is melanoma, carcinoma, or sarcoma.  
     
     
         6 . The method according to  claim 5 , wherein the cancer is cervical cancer.  
     
     
         7 . The method according to  claim 1 , wherein the converted cell represents cervical dysplasia.  
     
     
         8 . The method according to  claim 7 , wherein the dysplasia is squamous intraepithelial lesion (SIL), low squamous intraepithelial lesion (LSIL), high squamous intraepithelial lesion (HSIL), carcinoma in situ (CIS) or cancer.  
     
     
         9 . The method according to  claim 1 , comprising confirming the methylation marker gene, which comprises assaying for methylation of the common identified gene in the converted cell, wherein the presence of methylation in the promoter region of the common identified gene confirms that the identified gene is the marker gene.  
     
     
         10 . The method according to  claim 9 , wherein the assay for methylation of the identified gene is carried out by 
 (iv) identifying primers that span a methylation site within the nucleic acid region to be amplified,    (v) treating the genome of the converted cell with a methylation specific restriction endonuclease,    (vi) amplifying the nucleic acid by contacting the genomic nucleic acid with the primers, wherein successful amplification indicates that the identified gene is methylated, and unsuccessful amplification indicates that the identified gene is not methylated.    
     
     
         11 . The method according to  claim 10 , wherein the converted cell genome is treated with an isoschizomer of the methylation sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG-sites as a control.  
     
     
         12 . A method of identifying a converted cell comprising assaying for the methylation of the marker gene identified in  claim 1 .  
     
     
         13 . A method of diagnosing cancer or a stage in the progression of the cancer in a subject comprising assaying for the methylation of the marker gene identified using the method in  claim 1 .  
     
     
         14 . The method according to  claim 13 , wherein the cancer is cervical cancer.  
     
     
         15 . The method according to  claim 14 , wherein the marker gene is Nucleoporin 98 kDa, Selenoprotein X, 1, DKFZP4340047 protein, Zinc finger protein 324, Testis-specific kinase 2, Corin, serine protease, GLI-Kruppel family member GLI2, Spermidine/spermine N1-acetyltransferase, Scaffold attachement factor B, Leucine-rich repeats and calponin homology (CH) domain containing 4, Laminin, beta 2 (Laminin S), ATPase Na+/K+ transporting, beta 2 polypeptide, Tubulin, beta polypeptide, Aldehyde dehydrogenase 3 family, member B1, Leukocyte tyrosine kinase, Procollagen C endopeptiase enhancer, Protein tyrosine phosphatase, receptor type, U, TAF10 RNA polymerase II, TATA box binding protein (TBP)-associated factor 30 kDa, Fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome), DNA-damage-inducible transcript 3, ADCYAP1 (NT — 010859): Adenylate cyclase activating polypeptide 1 (pituitary); C10orf116 (NT — 030059): Chromosome 10 open reading frame 116; CCNA1 (NT — 024524): Cyclin A1; CCND2 (NT — 009759): Cyclin D2; EPHA5 (NT — 022778): EphA5; HOXA11 (NT — 007819): Homeo box A11; IGFBP4 (NT — 010755): Insulin-like growth factor binding protein 4; KIAA1467 (NT — 009714); LHX6 (NT — 008470): LIM homeobox 6; MAL (NT — 026970): Mal, T-cell differentiation protein; MRC2 (NT — 010783): Mannose receptor, C type 2; RASL12 (NT — 010194): RAS-like, family 12; RPL23AP7 (MGC70863, NT — 011526): SIMILAR TO RIBOSOMAL PROTEIN L23A; SLC30A3 (NT — 022184): Solute carrier family 30 (zinc transporter), member 3; TBX3 (NT — 009775): T-box 3 (ulnar mammary syndrome); VIM (NT — 077569): Vimentin; ZFHX1B (NT — 005058); ZNF486 (NT — 011295); CD34 (NT — 021877): CD34 antigen; CDC34 (NT — 011255): Cell division cycle 34; CTF1 (NT — 086679): Cardiotrophin 1; CX3CR1 (NT — 022517): Chemokine (C-X3-C motif) receptor 1; FDPS (NT — 004487): FARNESYL PYROPHOSPHATE SYNTHETASE (FPPS); GSTM4 (NT — 019273): Glutathione S-transferase M4; MYH7B (NT — 028392): MYOSIN, HEAVY POLYPEPTIDE 7B, CARDIAC MU; SEC61A2 (NT — 077569): Sec61 alpha 2 subunit ( S. cerevisiae ); STOML1 (NT — 010194): Stomatin (EPB72)-like 1; and THBD (NT — 011387): Thrombomodulin, and combinations thereof.  
     
     
         16 . A method of diagnosing high grade lesion of cervical dysplasia comprising assaying for methylation of a marker gene as follows: ADCYAP1 (NT — 010859): Adenylate cyclase activating polypeptide 1 (pituitary); C10orf116 (NT — 030059): Chromosome 10 open reading frame 116; CCNA1 (NT — 024524): Cyclin A1; CCND2 (NT — 009759): Cyclin D2; EPHA5 (NT — 022778): EphA5; HOXA11 (NT — 007819): Homeo box A11; IGFBP4 (NT — 010755): Insulin-like growth factor binding protein 4; KIAA1467 (NT — 009714); LHX6 (NT — 008470): LIM homeobox 6; MAL (NT — 026970): Mal, T-cell differentiation protein; MRC2 (NT — 010783): Mannose receptor, C type 2; RASL12 (NT — 010194): RAS-like, family 12; RPL23AP7 (MGC70863, NT — 011526): SIMILAR TO RIBOSOMAL PROTEIN L23A; SLC30A3 (NT — 022184): Solute carrier family 30 (zinc transporter), member 3; TBX3 (NT — 009775): T-box 3 (ulnar mammary syndrome); VIM (NT — 077569): Vimentin; ZFHX1B (NT — 005058); ZNF486 (NT — 011295); CD34 (NT — 021877): CD34 antigen; CDC34 (NT — 011255): Cell division cycle 34; CTF1 (NT — 086679): Cardiotrophin 1; CX3CR1 (NT — 022517): Chemokine (C-X3-C motif) receptor 1; FDPS (NT — 004487): FARNESYL PYROPHOSPHATE SYNTHETASE (FPPS); GSTM4 (NT — 019273): Glutathione S-transferase M4; MYH7B (NT — 028392): MYOSIN, HEAVY POLYPEPTIDE 7B, CARDIAC MU; SEC61A2 (NT — 077569): Sec61 alpha 2 subunit ( S. cerevisiae ); STOML1 (NT — 010194): Stomatin (EPB72)-like 1; and THBD (NT — 011387): Thrombomodulin or a combination thereof  
     
     
         17 . The method according to  claim 16 , wherein high grade lesion is intraepithelial lesion (HSIL), carcinoma in situ (CIS) or cancer.  
     
     
         18 . The method according to  claim 16 , wherein the dysplasia is observed in sample taken from scrape, biopsy, blood or urine.  
     
     
         19 . A method of diagnosing high grade lesion of cervical dysplasia comprising assaying for methylation of a marker gene as follows: ZNF324, TESK2, LTK, NUP98, TAF10, SAT, SEPX1, PCOLCE, VIM, DDIT3, LHX6, CCND2, ZFHX1B, TBX3, ADCYAP1, SAFB, RASL12, FGFR1, HOXA11, CCNA1, RPL23AP7.